Botta et al. Lipids in Health and Disease (2020) 19:156 https://doi.org/10.1186/s12944-020-01332-5 RESEARCH Open Access An adiponectin-S1P autocrine axis protects skeletal muscle cells from palmitate- induced cell death Amy Botta, Kazaros Elizbaryan, Parastoo Tashakorinia, Nhat Hung Lam and Gary Sweeney* Abstract Background: The prevalence of type 2 diabetes, obesity and their various comorbidities have continued to rise. In skeletal muscle lipotoxicity is well known to be a contributor to the development of insulin resistance. Here it was examined if the small molecule adiponectin receptor agonist AdipoRon mimicked the effect of adiponectin to attenuate palmitate induced reactive oxygen species (ROS) production and cell death in L6 skeletal muscle cells. Methods: L6 cells were treated ±0.1 mM PA, and ± AdipoRon, then assays analyzing reactive oxygen species (ROS) production and cell death, and intracellular and extracellular levels of sphingosine-1 phosphate (S1P) were conducted. To determine the mechanistic role of S1P gain (using exogenous S1P or using THI) or loss of function (using the SKI-II) were conducted. Results: Using both CellROX and DCFDA assays it was found that AdipoRon reduced palmitate-induced ROS production. Image-IT DEAD, MTT and LDH assays all indicated that AdipoRon reduced palmitate-induced cell death. Palmitate significantly increased intracellular accumulation of S1P, whereas in the presence of AdipoRon there was increased release of S1P from cells to extracellular medium. It was also observed that direct addition of extracellular S1P prevented palmitate-induced ROS production and cell death, indicating that S1P is acting in an autocrine manner. Pharmacological approaches to enhance or decrease S1P levels indicated that accumulation of intracellular S1P correlated with enhanced cell death. Conclusion: This data indicates that increased extracellular levels of S1P in response to adiponectin receptor activation can activate S1P receptor-mediated signaling to attenuate lipotoxic cell death. Taken together these findings represent a possible novel mechanism for the protective action of adiponectin. Keywords: Adiponectin, Sphingosine-1 phosphate, Skeletal muscle cells, Palmitate, ROS, Lipotoxicity, Cell death, AdipoRon Background under lipotoxic conditions reactive oxygen species (ROS) are Theprevalenceoftype2diabetesandobesityhascontinued produced, leading to increased lipid peroxidation, which in to rise and with this various comorbidities have become per- turn also leads to increased levels of cell death [6, 7]. It has vasive [1]. Skeletal muscle is a metabolically active tissue in been shown that addition of palmitate to skeletal muscle which there are high levels of mitochondria [2, 3]. Lipotoxi- cells leads to increased levels of superoxide [8, 9]. Previous city in skeletal muscle is well recognized as a contributor to research has shown that palmitate (PA) induced cell death is the development of insulin resistance [4, 5]. In addition, in part due to increased accumulation of distinct ceramide species [10, 11]. While increased levels of ceramide are con- * Correspondence: [email protected] sidered to be toxic, several metabolites of ceramide, such as Department of Biology, York University, Toronto, ON M3J 1P3, Canada © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Lipids in Health and Disease (2020) 19:156 Page 2 of 9 sphingosine-1 phosphate (S1P) are known to mediate benefi- 10% volume/volume (v/v) fetal bovine serum and 1% cial cellular responses, such as anti-apoptotic effects [12]. antibiotic/antimycotic solution (v/v, Wisent Inc., Saint- Lack of adiponectin action has now been implicated in Jean-Baptist, Quebec, Canada). Prior to passage and many disease states, perhaps most especially type 2 dia- seeding for experiments cells were grown to a maximum betes in obesity [13, 14]. Healthy individuals typically have of 80% confluency in 75cm2 flasks at 37 °C and 5% CO2. high concentrations of adiponectin within the circulation L6 cells were plated and left overnight and then were in- [15]. In both diabetes and obesity circulating levels of adi- cubated for 4 h with 0.5% BSA containing medium. ponectin are significantly reduced [16–18]. Although the Then 0.1 mM of PA dissolved in 3% bovine serum albu- bulk of circulating adiponectin derives from adipose tis- min (BSA) or BSA control was added. A subset of cells sue, previous research has in fact shown that skeletal was treated with either 35 μM AdipoRon (Cayman muscle can produce and secrete adiponectin [19, 20]. Adi- Chemical, Ann Arbor, Michigan, United States), 2.5 μM ponectin has been shown to regulate fatty acid metabol- sphingosine-1-phosphate (S1P, Sigma Aldrich, St. Louis, ism in muscle and can lead to increased fatty acid uptake Missouri, United States), 5 μM sphingosine kinase in- and decreased fatty acid synthesis [21, 22]. Previous re- hibitor II (SKI-II, Sigma Aldrich, St. Louis, Missouri, search has shown that under high-fat feeding conditions United States), or 5 μM 2-Acetyl-5-tetrahydroxybutyl in mice, adiponectin can improve insulin sensitivity and imidazole (THI, Sigma Aldrich, St. Louis, Missouri, prevent damage to skeletal muscle cells [23]. Since lower United States) as indicated. levels of adiponectin have been implicated in several meta- bolic disease states [24–26] there has been significant Analysis of intracellular ROS interest in the identification of small molecule adiponectin CellROX Green (Thermofisher Scientific, Waltham, receptor agonists. One such small molecule is AdipoRon, Massachusetts, United States) was utilized to detect ROS which has been shown to mimic adiponectin signaling production in live cells following manufacturers instruc- both in multiple cell types and in animal models [27, 28]. tions using an EVOS FL Auto 2 Cell Imaging System One important feature of adiponectin action, which could (Thermofisher Scientific, Waltham, Massachusetts, be beneficial during lipotoxic conditions, is that it stimulates United States). Additionally, for plate-based assays, 2′, the production of S1P, a signaling sphingolipid that is formed 7′-Dichlorofluorescin Diacetate (DCF-DA, Sigma Al- from the conversion of ceramide into sphingosine by cerami- drich, St. Louis, Missouri, United States) was utilized to dases and subsequent conversion of sphingosine into S1P by detect ROS as previously described [40]. sphingosine kinase [29, 30]. Adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2), both have intrinsic ceramidase activity which Assays to measure cell death is activated after binding with adiponectin or receptor agonists Image-IT DEAD Green Viability Stain (Thermofisher [31, 32]. This increase in ceramidase activity leads to increased Scientific, Waltham, Massachusetts, United States) was conversion of ceramide into S1P, thereby reducing the buildup utilized to detect cell death after 24 h incubation follow- of ceramides and related lipotoxic molecules [31–34]. Previous ing manufacturers instructions using a Nikon ECLIPSE research has shown that S1P plays an important role in skel- Ti2 (Nikon, Tokyo, Japan). Using a kit, the release of etal muscle regeneration [35, 36]. Additionally, increased levels lactate dehydrogenase (LDH) was determined (G-Biosci- of S1P have been linked to decreased insulin resistance and ences, St. Louis, Missouri, United States). Briefly, 25 ul of cell death [37–39]. However, whether adiponectin-mediated media was transferred to a separate 96-well plate. 25 ul of increases in S1P action are required to confer anti-lipotoxic ef- reconstituted substrate mix was then added and the plate fects in skeletal muscle cells has not been fully elucidated. was incubated for 30 min. Following which, 25 ul of stop Here rat L6 skeletal muscle cells treated with palmitate solution was added and the absorbance was then mea- with or without AdipoRon were used to determine intra- sured at 490 nm. The 3-(4,5-dimethylthiazol-2-yl)-2,5-di- cellular and extracellular S1P levels, ROS production phenyltetrazolium bromide (MTT) assay (Sigma Aldrich, and cell death. A subset of cells were also treated cells St. Louis, Missouri, United States) was conducted by incu- with S1P and pharmacological inhibitors to enhance or bating cells with 5 mg/ml MTT in PBS for 1 h at 37 °C reduce S1P production. The findings provide new know- and 5% CO2. After incubation cells were washed with ledge on the mechanistic role of S1P in mediating bene- PBS. DMSO (Sigma Aldrich, St. Louis, Missouri, United ficial effects of adiponectin in skeletal muscle cells. States) was then added to dissolve the precipitated forma- zan. The absorbance was then measured at 570 nm. Methods Cell culture Measurement of S1P levels Rat L6 skeletal muscle myoblasts were incubated in L6 cells were plated in 6 well plates and were treated alpha modified Eagle medium (α-MEM, Wisent Inc., with either PA or BSA with or without the addition of Saint-Jean-Baptist, Quebec, Canada) supplemented with 35 uM AdipoRon. After 24 h the media (1 ml) was Botta et al. Lipids in Health and Disease (2020) 19:156 Page 3 of 9 removed from the cells and placed in a separate tube.